Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle

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   FIG 1

   Differential infection of epithelial cells and mouse MΦ by genetically defined strains of IAV that differ only in HA gene expression. Monolayers of LA-4 epithelial cells (top panels) and PEC MΦ (bottom panels) were infected with an MOI of 5 PFU/cell of RG-PR8-HA or RG-Braz-HA for 1 h at 37°C. Excess virus was removed by washing, and the cells were then fixed at either 2 h or 8 h postinfection and stained by immunofluorescence for expression of newly synthesized viral NP (green) as described in Materials and Methods. DAPI staining (blue) was performed to visualize cell nuclei. (A) Viral NP⁺ cells are visible at 8 h postinfection in epithelial cells infected with either RG-PR8-HA or RG-Braz-HA and only in MΦ infected with RG-Braz-HA. Magnification, ×20. (B) Monolayers of epithelial cells (MDCK and LA-4) and mouse MΦ (BAL fluid MΦ, PEC MΦ, and RAW264.7 cells) were infected with RG-PR8-HA or RG-Braz-HA as described above and then fixed at 8 h postinfection before staining by immunofluorescence to detect newly synthesized viral NP. Data represent the mean percent infection (± standard error of the mean [SEM] [error bar]) from no less than four independent fields per chamber and are representative of at least three separate experiments.
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   FIG 2

   IAV-infected epithelial cells, but not mouse MΦ, support productive viral infection. (A) Monolayers of epithelial cells (MDCK and LA-4 cells) and mouse MΦ (PEC and RAW264.7 cells) were incubated with an MOI of 5 PFU/cell of RG-Braz-HA for 1 h, washed extensively to remove excess virus, and then cultured. At 2 and 24 h postinfection, cell culture supernatants were removed and clarified by centrifugation, and titers of infectious virus were determined by plaque assay on MDCK cells. Data represent the mean fold increase in viral titer between 2 h and 24 h postinfection (± SEM), pooled from three to five independent experiments. (B) Cell monolayers were incubated with an MOI of 0.01 PFU/cell of RG-Braz-HA for 1 h, washed extensively to remove excess virus, and then cultured in serum-free medium supplemented with 1 μg/ml TPCK-treated trypsin. At 2, 24, and 48 h postinfection, cell culture supernatants were removed and clarified by centrifugation, and titers of infectious virus were determined by plaque assay on MDCK cells. Data show the mean virus titers from triplicate samples (± SEM) in PFU per milliliter and are representative of at least two independent experiments.
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   FIG 3

   Mouse MΦ, but not epithelial cells, differ in susceptibility and responsiveness to different IAV strains through the induction of host antiviral factors. Monolayers of mouse LA-4 epithelial cells and PEC MΦ were infected with an MOI of 5 PFU/cell of RG-PR8-HA or RG-Braz-HA or mock infected for 1 h at 37°C. Monolayers were washed to remove excess virus, and then cells were lysed, and total RNA was extracted at 2 h or 6 h postinfection. The levels of host antiviral genes Ifit1, Ifit3, and Eif2ak2 (top panels), as well as Ifih1, Mx1, and Isg20 (bottom panels) were determined by qRT-PCR using TaqMan gene expression assays as described in Materials and Methods. For each gene of interest, relative gene expression (± 1 standard deviation [SD]) was determined using the 2^−ΔCT method described in Materials and Methods, and data were pooled from three independent experiments.
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   FIG 4

   Both RG-PR8-HA and RG-Braz-HA bind to epithelial cells and mouse MΦ; however, inhibition of the viral NA results in enhanced infection of mouse MΦ by RG-PR8-HA. (A) Epithelial cells (MDCK) and mouse MΦ (RAW264.7) were incubated with 10 μg/ml of purified RG-PR8-HA and RG-Braz-HA or no virus (mock infected) for 30 min on ice. After the cells were washed, virus bound to the cell surface was detected using anti-NA polyclonal rabbit sera in conjunction with goat anti-rabbit Ig–FITC conjugate, followed by flow cytometry. (i) Representative histograms show binding of RG-PR8-HA and RG-Braz-HA to epithelial cells and MΦ. Staining of mock-infected cells are displayed as solid gray histograms. (ii) Data are expressed as the mean fold change (± SEM) in the geometric means of RG-PR8-HA or RG-Braz-HA bound to the cell surface relative to the geometric mean of the no-virus (mock) control. The data show pooled triplicate samples from three independent experiments. There was no significant difference (n.s) in the level of RG-PR8-HA compared with RG-Braz-HA bound to epithelial cells (P = 0.16) or MΦ (P = 0.45) (two-tailed Student's t test). (B) Monolayers of mouse MΦ (PEC) were infected with 10⁷ PFU (MOI of 50 PFU/cell) of RG-PR8-HA or 10⁶ PFU (MOI of 5 PFU/cell) of RG-Braz-HA for 1 h at 37°C in the presence or absence (control) of 10 nM zanamivir. Prior to infection, cells were also treated with 10 mg/ml mannan or 5 mg/ml asialofetuin (ASF) or mock treated to block CLR-virus interactions. Monolayers were then washed and incubated for a further 6 to 7 h in the presence or absence of zanamivir with either mannan or ASF or mock treatment. Cells were fixed and stained by immunofluorescence to detect expression of newly synthesized viral NP. Data represent the mean percent infection (± SEM) from no less than four independent fields per chamber and are representative of two independent experiments. Values that were significantly different (P < 0.05 by one-way ANOVA with Tukey's posthoc analysis) are indicated by bar and asterisk. Values that were not significantly different (P > 0.05) are indicated by a bar and n.s.
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   FIG 5

   Viral mRNA and vRNA for each gene segment is produced in mouse MΦ and epithelial cells infected with RG-Braz-HA. Monolayers of mouse MΦ (PEC MΦ) and epithelial cells (MDCK and LA-4 cells) were incubated with an MOI of 5 PFU/cell of RG-Braz-HA for 1 h at 37°C, washed, and incubated further until total RNA was extracted 2 and 6 h postinfection. The levels of vRNA and mRNA for each IAV gene were determined via qRT-PCR as described in Materials and Methods. Data are expressed as the mean copy number (± SEM) at 6 h postinfection for each IAV gene, pooled from three independent experiments. The copy number at 2 h postinfection for each gene is represented as a horizontal line on each bar.
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   FIG 6

   Expression of IAV proteins in epithelial cells and MΦ infected with RG-Braz-HA. (A) Monolayers of mouse MΦ (RAW264.7 cells) and epithelial cells (LA-4 and MDCK cells) were incubated with an MOI of 5 PFU/cell of RG-Braz-HA or no virus (−) for 1 h at 37°C and washed to remove excess virus. At 2 h and 16 h postinfection, total cell lysates were prepared, resolved by SDS-PAGE under reducing conditions, and transferred to a PVDF membrane. IAV proteins from infected cells were detected by Western blotting, using antibodies specific for each viral protein. β-Actin (approximately 42 kDa) was monitored to ensure equivalent loading between samples. Proteins of the predicted molecular mass were detected using anti-IAV antibodies specific for NP (56 kDa), M1 (27 kDa), M2 (doublet, approximately 11 kDa), NS1 (26 kDa), and NS2 (14 kDa) in epithelial cells and MΦ at 16 h postinfection. Data are representative of at least three independent samples. (B) Monolayers of mouse MΦ (RAW264.7 cells) and epithelial cells (LA-4 and MDCK cells) were incubated with an MOI of 5 PFU/cell of RG-Braz-HA or no virus (mock) for 1 h at 37°C and washed to remove excess virus. At 2 h and 8 h postinfection, viral HA and viral NA expressed on the cell surface was detected using HA-specific and NA-specific antibodies, respectively, in conjunction with flow cytometry. Data are representative of duplicate experiments.
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   FIG 7

   Virus budding from RG-Brazil-HA-infected epithelial cells but not mouse MΦ. Monolayers of mouse MΦ (PEC) and epithelial cells (MDCK cells) were incubated with an MOI of 5 PFU/cell of RG-Braz-HA for 1 h at 37°C, washed to remove excess virus, and cultured. At 16 h postinfection, cells were fixed and examined by TEM as described in Materials and Methods. Virions and densely stained particles budding from the plasma membrane of epithelial cells (indicated by black arrows), but not from mouse MΦ, can be observed.

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